an engineered monolignol 4-o-methyltransferase depresses lignin biosynthesis and confers novel...

An Engineered Monolignol 4-O-Methyltransferase DepressesLignin Biosynthesis and Confers Novel Metabolic Capabilityin ArabidopsisC W OA

Kewei Zhang,a,1 Mohammad-Wadud Bhuiya,a,1,2 Jorge Rencoret Pazo,b Yuchen Miao,a,3 Hoon Kim,b John Ralph,b

and Chang-Jun Liua,4a Department of Biology, Brookhaven National Laboratory, Upton, New York 11973bDepartment of Biochemistry, The Wisconsin Bioenergy Initiative and the Department of Energy Great Lakes Bioenergy ResearchCenter, University of Wisconsin, Madison, Wisconsin 53706

Although the practice of protein engineering is industrially fruitful in creating biocatalysts and therapeutic proteins,applications of analogous techniques in the field of plant metabolic engineering are still in their infancy. Lignins arearomatic natural polymers derived from the oxidative polymerization of primarily three different hydroxycinnamyl alcohols,the monolignols. Polymerization of lignin starts with the oxidation of monolignols, followed by endwise cross-coupling of(radicals of) a monolignol and the growing oligomer/polymer. The para-hydroxyl of each monolignol is crucial for radicalgeneration and subsequent coupling. Here, we describe the structure-function analysis and catalytic improvement of anartificial monolignol 4-O-methyltransferase created by iterative saturation mutagenesis and its use in modulating lignin andphenylpropanoid biosynthesis. We show that expressing the created enzyme in planta, thus etherifying the para-hydroxyls oflignin monomeric precursors, denies the derived monolignols any participation in the subsequent coupling process,substantially reducing lignification and, ultimately, lignin content. Concomitantly, the transgenic plants accumulated denovo synthesized 4-O-methylated soluble phenolics and wall-bound esters. The lower lignin levels of transgenic plantsresulted in higher saccharification yields. Our study, through a structure-based protein engineering approach, offers a novelstrategy for modulating phenylpropanoid/lignin biosynthesis to improve cell wall digestibility and diversify the repertories ofbiologically active compounds.

INTRODUCTION

Lignin is a major biopolymer in secondary cell walls of terrestrialplants. However, its presence presents a formidable obstacle tothe efficient use of cellulosic fibers in agricultural and industrialapplications, particularly the conversion of cellulosic biomass toliquid biofuels (Weng et al., 2008). Although lignin biosynthesishas been studied for decades and considerable efforts havebeen dedicated to modulating lignin content or its compositionto more economically use cell wall fibers, novel biotechnology/methodology is still required for efficiently manipulating plantlignification.

Lignin is generally considered to arise from three monolignols,p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, whichdiffer in their degree of meta-methoxylation (methoxylation orthoto the phenol) (see Supplemental Figure 1 online). After theirsynthesis in the cytosol, they are transported across the cellmembrane and deposited into the cell wall, where they are oxi-dized and polymerized to yield p-hydroxyphenyl (H), guaiacyl (G),and syringyl (S) units, respectively, when incorporated into thelignin polymer. The composition of lignin varies between differentplant species and tissues. Lignin from gymnosperms and otherlower vascular plants is rich in G-units and contains low amountsof H-units, and dicot lignin is mainly composed of G- and S-units(Weng and Chapple, 2010). The G-unit is monomethoxylated (viamethylation of the 3-hydroxyl group in caffeyl intermediates),whereas the S-unit is dimethoxylated (via additional methylationof the 5-hydroxyl moieties in 5-hydroxyguaiacyl intermediates).The ratio of S:G varies from 0:100 to nearly 100:0, resulting inalterations in lignin structures that dictate the degree of lignincondensation, reactivity, and recalcitrance to delignification(Boerjan et al., 2003; Ralph et al., 2004b).The monolignols are synthesized from a branch of the phenyl-

propanoid pathway, where ;10 catalytic enzymes constitute a lin-ear pathway converting the aromatic amino acid L-Phe to the threehydroxycinnamyl alcohols (see Supplemental Figure 1 online). Inmembers of the Brassicaceae, including Arabidopsis thaliana, thebiosynthetic intermediates, hydroxycinnamaldehydes, on thispathway can be diverted to yield methanolic soluble phenolic

1 These authors contributed equally to this work.2 Current address: Conagen, 1005 North Warson Road, St Louis, MO63132.3 Current address: Henan University, Jinming Road, Kaifeng, HenanProvince 475004, China.4 Address correspondence to [email protected] author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Chang-Jun Liu ([email protected]).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.112.101287

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esters, primarily sinapoyl malate in leaves, and sinapoyl cholinein seeds, which act as a UV screen and pest deterrent, re-spectively (Landry et al., 1995; Milkowski et al., 2004; Nair et al.,2004) (see Supplemental Figure 1 online).

Earlier studies revealed that monolignols undergo dehy-drodimerization or polymerization in an in vitro horseradishperoxidase–hydrogen peroxide (H2O2) system to form syntheticlignins (i.e., dehydrogenation polymers), with most of thestructural features found in plant lignins (Freudenberg, 1959).Consequently, it is envisioned that lignin polymerization occursvia one-electron oxidation of phenols by oxidative enzymes(peroxidases/laccases) to yield resonance-stabilized phenolicradicals; subsequently, the coupling of phenoxy radicals witheach other or, more importantly, with the growing polymer,

forms lignin (Figure 1A) (Freudenberg, 1968; Boerjan et al., 2003;Ralph et al., 2004b; Davin et al., 2008). The cross-coupling ofphenoxy radicals from the three monolignols (i.e., p-coumaryl,coniferyl, and sinapyl alcohols) within the plant cell wall gen-erates different lignin interunit linkages, including the most fre-quent b-aryl ether (8–O–4) units and less frequent b–b (8–8) andb–5 (8–5) units; coupling between preformed oligomer/polymerunits forms 5–5- and less frequent 5–O–4-linked units (Boerjanet al., 2003; Ralph et al., 2004b) (Figure 1A). In this oxidativecoupling process, an unsubstituted phenol, the free para-hydroxylof the monolignol, was critically implicated in generating phenoxyradical intermediates and in forming different types of ligninsubunits (Harkin, 1967, 1973; Ralph et al., 2004b, 2009). There-fore, we rationalized that a chemical modification, for example,

Figure 1. Schema of the Proposed Oxidative-Dehydrogenation and Phenoxy-Coupling, and the Reactions Catalyzed by Phenolic O-Methyl-transferases.

(A) The active radical intermediates are generated from the monolignol (coniferyl alcohol) by oxidation of its phenol. The phenoxy radicals coupletogether or, more importantly, with the growing lignin oligomer/polymer yielding units with different interunit linkages in lignins.(B) The 4-O-methylation of phenylpropenes catalyzed by IEMT.(C) The engineered monolignol 4-OMT (MOMT) (in gray box); note the structural analogy between the monolignol coniferyl alcohol and the phenyl-propene isoeugenol.[See online article for color version of this figure.]

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the methylation of the phenol (the para-hydroxyl), would preventradical generation and deny the derived monolignols any partici-pation in the subsequent coupling process, thus reducing thequantity of lignin produced.

Monolignol biosynthesis requires the activities of two types ofO-methyltransferases, the caffeoyl CoA 3-O-methyltransferase(EC. 2.1.1.104), which specifically catalyzes the methylation ofcinnamoyl CoA conjugates (Franke et al., 2002; Boerjan et al.,2003), and a caffeate/5-hydroxyferulate 3/5-O-methyltransferase(COMT) (EC. 2.1.1.6), which preferentially methylates hydroxy-cinnamaldehydes or hydroxycinnamyl alcohols (Inoue et al., 2000;Parvathi et al., 2001) (see Supplemental Figure 1 online). However,both enzymes exclusively methylate the meta-hydroxyls, andnone of them display activity toward 4-O-methylation (Parvathiet al., 2001; Bhuiya and Liu, 2010). However, an O-methyl-transferase in fairy fans (Clarkia breweri), namely, isoeugenolO-methyltransferase (IEMT) (EC. 2.1.1.146), catalyzes the 4-O-methylation of volatile compounds isoeugenol and eugenol(Wang and Pichersky, 1998, 1999) (Figure 1B). These allyl/propenylphenols are structural analogs of monolignols, differingonly in their propanoid tail (Figure 1B). IEMT exhibits strict sub-strate specificity, showing none or only negligible activity towardmonolignols (Bhuiya and Liu, 2010). At the amino acid level, itshares more than 83% identity with lignin COMT from the samespecies. Phylogenetic analysis suggests it evolved from the pa-rental enzyme COMT through functional diversification (Wang andPichersky, 1998). Previously, Wang and Pichersky showed thatswapping sequence fragments of IEMT and COMT, or convertingsome residues around their putative active sites, invariablyand simultaneously alters their substrate preference and regio-selectivity of methylation (i.e., the mutations change the activity ofIEMT from the 4-O-methylation of allyl/propenylphenols to the3/5-O-methylation of caffeic acid and vice versa), although thecatalytic efficiency of the resulting variants was greatly com-promised (Wang and Pichersky, 1998, 1999). The evolutionaryrelationship of IEMT with the lignin biosynthetic enzyme and itsfunctional amenability suggest the potential of using IEMTas template to create a novel enzyme conferring the ability to4-O-methylate monolignols through protein engineering.

Protein engineering has proven a powerful strategy for en-hancing the stability, activity, and/or selectivity of enzymes.During the last decade, the practice of rational design andlaboratory-directed protein evolution has become firmlyestablished as a versatile tool in biochemical research byenabling molecular evolution toward desirable phenotypes orfor the detection of novel structure-function interactions. Thisstrategy has led to a number of successes in creating novelbiocatalysts for industrial applications. However, the practice ofprotein engineering for plant metabolites is rare. The mostsuccessful cases in altering the properties of plant secondarymetabolic enzymes are on sesquiterpene synthases. By sys-tematically exploring the relationship between sequence andfunction, Yoshikuni et al. (2006) identified and mutated a set ofhot spots near the active site of sesquiterpene synthases thatresults in biocatalysts selectively catalyzing the formation of aspectrum of sesquiterpene products). Furthermore, O’Maille et al.(2008) systematically investigated the relationship between acombination of nine naturally occurring amino acid substitutions

and the resulting sesquiterpene product spectra. However, theapplication of the resulting catalysts was not attempted in plantafor diversifying plant metabolism.Based on the IEMT homology modeling analysis and using the

available COMT crystal structure (Zubieta et al., 2002), we pre-viously identified a subset of evolutionarily useful hot spot res-idues in the active site of IEMT (Bhuiya and Liu, 2010). Sitesaturation mutagenesis yielded a few single to triple mutantvariants that confer the ability to regiospecifically methylate thepara-hydroxyl of monolignols; we termed them monolignol 4-O-methyltransferases (MOMTs) (Bhuiya and Liu, 2010). Althoughthe triple mutant variants displayed considerable in vitro activityin etherifying monolignols, when overexpressed in planta theyessentially did not result in any of the anticipated effects onphenylpropanoid and lignin biosynthesis. To further improve thecatalytic properties of MOMT to obtain a physiologically effec-tive enzyme, in this study, we explored the structural basis of thecreated MOMT variant for its regiospecific methylation by de-termining the x-ray crystal structure of a triple mutant variant;subsequently we refined its catalytic efficiency via additionaliterations of saturation mutagenesis. We then expressed thecreated artificial enzyme in Arabidopsis, resulting in a substantialreduction of the lignin quantity in the cell wall and an increase inthe yield of biomass saccharification concomitant with the pro-duction of novel phenylpropanoid metabolites.

RESULTS

Expressing the Early Set of MOMT Mutant Variants DoesNot Affect Phenylpropanoid Metabolism in Planta

Our previous study created a subset of single (E165F) to triple(T133L-E165I-F175I) mutant variants of IEMT with significantcatalytic efficiencies (kcat/Km of up to 1500 M21 s21) for methyl-ating the para-hydroxyl of monolignols (Bhuiya and Liu, 2010) (seeSupplemental Table 1 online). We expressed both the single andtriple mutant variants in either tobacco (Nicotiana tabacum) orArabidopsis, driven by either the constitutive double-35S pro-moter or the bean (Phaseolus vulgaris) phenylalanine ammonialyase-2 (PAL2) promoter, which controls the expression of the firstkey enzyme in the phenylpropanoid pathway in vascular tissues(Cramer et al., 1989; Reddy et al., 2005). However, the resultingtransgenic plants showed essentially no detectable change in thelignin content of cell wall and no change in the accumulation ofother soluble phenolics. Among the possible reasons for thefailure of the created enzymes to function in planta, one obviousobstacle probably lies in less than optimal catalytic efficiency.

Crystal Structure of MOMT3 and Improvement of ItsCatalytic Efficiency

To improve the MOMT mutant enzymatic properties and to ex-plore the structural basis for the altered substrate specificity ofthose variants (Bhuiya and Liu, 2010), we determined the crystalstructure of a triple mutant variant, MOMT3 (T133L-E165I-F175I), complexed with the substrate coniferyl alcohol, andS-adenosyl homocysteine (SAH), the product of methyl donor

Modulating Lignin Synthesis with 4-OMT 3 of 18



S-adenosyl Met (SAM) (Figure 2A). The initial structure modelwas solved by molecular replacement using the well-definedcaffeic acid 3-O-methyltransferase structure (Zubieta et al.,2002) and refined to a resolution of 2.4 Å (see Supplemental Table2 online). Similarly to the previously characterized plant phenolicO-methyltransferases (Zubieta et al., 2002; Louie et al., 2010),MOMT3 forms a homodimer, and each monomer consists ofa large C-terminal catalytic domain involved in SAM/SAH andphenolic substrate binding and a small N-terminal domain thatprimarily mediates dimerization (Figure 2A). The N-terminal do-main also contributes to the formation of the active site of thedyad molecule by providing residues (Met-26 and Ser-30) liningthe back wall of the substrate binding cavity and helping enclosethe recognition surface (Figures 2A and 2B). As depicted, coniferylalcohol bound in the active site is tethered by three hydrogenbonds formed via the g-hydroxyl of its propanoid tail with Ser-30of the dyad molecule of MOMT3 dimer and its 4-hydroxyl (phenol)with the side chains of Asn-327 and Asp-273 (Figure 2B). More-over, its meta-methoxy moiety is buried in a hydrophobic pocketconstituted by Leu-133 and Ile-165 (as established via mutationsof Thr-133 and Glu-165 of IEMT), as well as by Leu-139, Phe-179,and Tyr-326 (Figure 2B). These H-bond linkages, the hydrophobicinteractions, and probably the steric effect generated by themutation (e.g., Thr-133 to Leu) enable monolignol bound in theactive site with its para-hydroxyl proximal to both the methyl-donor and the catalytic base His-272 for transmethylation.Based on the crystal structure information, 20 amino acid

residues directly in contact with, or proximal to, the bound con-iferyl alcohol were unequivocally identified (see SupplementalFigure 2 online). To further enhance the catalytic efficiency ofMOMT for 4-O-methylation of monolignols, we performed an it-erative saturation mutagenesis of those identified residues(Phe-130, Ala-134, Val-138, Leu-139, Phe-157, Tyr-161, Asn-164,Phe-166, Tyr-168, His-169, His-173, Phe-179, Met-183, Asn-186,Thr-319, Leu-322, Met-323, Ala-325, Tyr-326, and Asn-327) usingthe created triple mutant variant MOMT3 (T133L-E165I-F175I) asthe template. This allowed us to screen out several mutant var-iants that bear an additional substitution(s) primarily at Tyr-161,Phe-166, or His-169, besides the three original mutations (Thr-133L, Glu-165I, and Phe-175I) of the MOMT3 parental enzyme(Figure 2C; see Supplemental Table 1 online). These tetra- andpenta-mutants exhibited higher enzymatic activity than the pa-rental MOMT3. Kinetics determination revealed that the catalyticefficiency (kcat/Km) of the mutants T133L-E165I-F175I-H169F(MOMT4) to both coniferyl alcohol and sinapyl alcohol reached upto 4000 (M21 s21), an ;200-fold increase over those of the IEMTwild-type enzyme (Figure 2C; see Supplemental Table 1 online).Such catalytic properties are comparable to those of the nativephenolic O-methyltransferases (Inoue et al., 2000; Parvathi et al.,2001). The MOMT4 enzyme predominantly reacted with coniferylFigure 2. Structure and Catalytic Efficiency of the Engineered MOMTs.

(A) The tertiary structure of the fashioned MOMT3 mutant variant.MOMT3 forms a dimer with an interface contributed by the small N-terminal domains from dyad molecules. Two chains are presented ingreen and cyan. The phenolic substrate (coniferyl alcohol) bound in theactive site is depicted in magenta, and the methyl donor product SAH iscolor coded as C in yellow, N in blue, and O in red.(B) A close-up view of the active site of MOMT3 in complex with thesubstrate coniferyl alcohol and the product of the methyl donor SAH. The

three amino acid substitutions are labeled in red. The image is acquiredfrom chain B of the dimer.(C) The catalytic efficiency (kcat/Km) of the created MOMT variants inmethylating coniferyl alcohol and sinapyl alcohol. For the detailed kineticparameters, see Supplemental Table 1 online.









and sinapyl alcohols and their corresponding aldehydes butshowed negligible activity toward p-coumaryl alcohol, its alde-hyde, and caffeyl alcohol, which lack the meta-O-methyl moiety(Table 1). This highlights the importance of the observed in-teraction of the meta-methoxyl of a monolignol with the hydro-phobic residues constituted by the mutations in the active site ofMOMT (as seen in Figure 2B) for constraining the proper orien-tation of the substrate to ensure efficient 4-O-methylation. Whena broader range of potential substrate phenolics was tested, thecreated enzyme showed only negligible activity on ferulic andsinapic acids, and none for feruloyl- and sinapoyl-CoAs and si-napoyl esters (sinapoyl glucose and malate) (Table 1), pointing outits relatively narrow substrate specificity.

4-O-Methylation of Monolignols Eliminates TheirOxidative Coupling in Vitro

To reevaluate the effect of substitution of the para-hydroxyl ofmonolignols on their coupling and cross-coupling propensities,we produced 4-O-methylated coniferyl and sinapyl alcoholsusing the created MOMT4 and subjected them to the peroxidase-catalyzed dehydrogenative polymerization. As expected, and insharp contrast with conventional monolignols (see SupplementalFigures 3A and 3B online), no dimeric or oligomeric products fromthe 4-O-methyl-monolignols were detected (see SupplementalFigures 3C and 3D online), confirming that methylation of thephenol (para-hydroxyl) indeed deprives the oxidative de-hydrogenation and the subsequent coupling of the modifiedcompounds in vitro.

To examine whether the 4-O-methyl-monolignol interfereswith the oxidative dehydrogenation and coupling of conven-tional lignin monomers, we first incubated an equal amount ofconiferyl alcohol and 4-O-methyl-coniferyl alcohol in the pres-ence of horseradish peroxidase and H2O2 and found that es-sentially the same amount and types of oxidative couplingproducts were produced as those derived from coniferyl alcoholalone (see Supplemental Figure 4A online). This suggests that4-O-methylation of the monolignol prevents the participation ofonly the modified compound into the coupling or polymerizationprocess and does not impair the activity of oxidative polymeri-zation of conventional monolignols. For more comprehensiveanalysis, we mixed a fixed concentration of coniferyl alcoholwith a series of concentrations of 4-O-methyl-coniferyl alcoholin the presence of peroxidase and H2O2 and quantified the majoroxidative dimeric products. Compared with those derivedfrom the polymerization reaction of coniferyl alcohol alone, thequantity of the detected coupling products in all the reactionscoincubating both conventional and 4-O-methylated mono-lignols did not show substantial changes (see SupplementalFigure 4B online), confirming that 4-O-methylated monolignolsdo not inhibit or compete with the peroxidase-catalyzed oxida-tive dehydrogenation of conventional monolignols.

Expressing MOMT4 in Arabidopsis Lowers LigninContent in the Cell Walls

To explore the potential effects of 4-O-methylation of mono-lignols on lignin polymerization in planta, we overexpressed

MOMT4 (T133L-E165I-F175I-H169F) in Arabidopsis. A loss-of-function IEMT variant (E165R) (Bhuiya and Liu, 2010) was in-cluded as the control. This E165R variant with substitution ofa Glu residue by a bulkier Arg that appreciably projects into theenzyme active site completely abolished catalytic activity(Bhuiya and Liu, 2010); therefore, it serves an ideal transgenicempty vector control. A bean PAL2 promoter (Reddy et al., 2005)was used to drive the transgenes (Figures 3A to 3C). The T2transgenic lines and their progenies were used for histochemicaland chemical compositional analyses. In MOMT4 overexpressionlines, cross sections of the basal internode of inflorescent stems(10 weeks old), under epifluorescence microscopy, exhibitedweaker autofluorescence and fewer numbers of fluorescent cellswithin their interfascicular fibers and xylem bundles, comparedwith those of the control plants (Figures 3D and 3E). Further-more, the conventional Wiesner (phloroglucinol-HCl, whereina violet-red color is indicative of lignins) and Mӓule (which stainssyringyl-containing lignins red) histochemical stains (Nakanoand Meshitsuka, 1992) exhibited a less intense coloration in thevasculatures of MOMT4 overexpression lines than in the con-trols (Figures 3F to 3I). These data point to a reduction in lignincontent in the cell walls of the MOMT transgenic lines. To assessthe quantitative alteration in lignin deposition as a consequenceof the expression of MOMT4, we measured the total lignincontent of 10-week-old T2 generation transgenic plants usingthe acetyl bromide method (Iiyama and Wallis, 1990; Changet al., 2008). Lignin content in all MOMT4 transgenic plants wasreduced, and the maximum reduction reached;24% in trans-genic line MOMT4-3, compared with the control plants (Table 2).To evaluate the alteration of lignin composition and, to some

extent, structure in the MOMT expression lines, we analyzed theproducts of analytical thioacidolysis by gas chromatography.Thioacidolysis primarily cleaves b–O–4-ether linkages inter-connecting the lignin subunits and therefore enables the directdetermination of the monomeric composition of noncondensedlignin (Lapierre, 1993). Calculating the yield of both the guaiacyland syringyl subunits on the basis of the cell wall dry mass re-vealed a significant reduction in lignin-derived monomers fromthe MOMT transgenic lines compared with the controls (Table2), which is consistent with the decrease in total lignin in theircell walls. However, the ratio of syringyl to guaiacyl units dis-played only slight alteration in the overexpression plants (Table2), suggesting that the structure of lignin in the MOMT over-expression lines is not modified drastically, although the quan-tity of total lignin was reduced. For a more detailed investigationinto the effects on lignin structure, whole-cell wall gel samples inDMSO-d6/pyridine-d5 and cellulolytic enzyme lignins (CELs)were further prepared from both the control and overexpressionlines and analyzed by NMR. The aromatic regions of two-dimensional (2D) 13C–1H correlation (heteronuclear single quan-tum coherence [HSQC]) NMR spectra of the equal amount of cellwall samples from overexpression and control lines showed thatthe signals of both G- and S-units were much lower in the cellwall of overexpression lines (Figure 4A; see Supplemental Figure5 online), confirming the reduction of total lignin, but the signalsof the p-hydroxyphenyl (H) unit remained the same or were onlyslightly reduced in overexpression lines, suggesting that MOMTmodifies predominantly the precursors for guaiacyl and syrigyl











lignin in vivo, which is consistent with its in vitro activity (Table1). Furthermore, the HSQC spectra of both aliphatic side chainand aromatic regions of prepared lignin (CEL) samples exhibitedno dramatic differences between the overexpression and con-trol lines, except for the slight increase of the relative amount ofH-subunit due to the decrease of G- and/or S-units in the lignin(Figure 4B; see Supplemental Figure 6 online). As expected, in2D-HSQC NMR spectra, no 4-O-methylated guaiacyl or syringylstructures as the end units were identified (Figure 4B; seeSupplemental Figure 6 online), confirming that aromatics with-out a free phenol (the para-hydroxyl) for initiating radical gen-eration have no bypass way of entering into polymerization.Together, these data imply that the 4-O-methylation of mono-lignols by MOMT reduced the incorporation of the modifiedlignin precursors (primarily for G- and S-units) into the polymersbut that the polymers produced were structurally normal re-garding their linkages.

4-O-Methylation of a Monolignol Does Not Impair ItsTransport across the Cell Membrane

To examine whether the decline of lignin polymerization in vivo isdue to the potential deprival of 4-O-methylated compoundsfrom their deposition into the cell wall, we prepared the invertedmembrane vesicles from wild-type Arabidopsis rosette leavesand primary bolting tissues (Miao and Liu, 2010) using anaqueous polymer two-phase partitioning system (Larsson et al.,1994). The quality of the prepared membrane fraction wasmonitored by measuring the vanadate-inhibited activity of theplasma membrane marker enzyme H+-ATPase. As depicted inFigure 5A, the detected ATP-dependent hydrolytic activity of theprepared inside-out vesicles was largely repressed with the ef-fective plasma membrane ATPase inhibitor, sodium vanadate(Gallagher and Leonard, 1982), suggesting the majority of the

inside-out membrane fraction represents plasma membrane.We then incubated the prepared inside-out vesicles with 4-O-methyl-coniferyl alcohol. In the presence or absence of ATP, theuptake of the 4-O-methyl monolignol by the inverted vesicles iscomparable to, or even higher than, that of conventional mono-lignols, indicating that 4-O-methylation did not abrogate thecompound’s transport across cell membranes (Figure 5B).Moreover, while the transport activity of the membrane vesiclesto coniferyl alcohol was greatly impaired in the absence of ATP,transport of the 4-O-methyl monolignol remained largely un-changed, implying that 4-O-methylation enhances passivediffusion, presumably due to an increase in the compound’s lipo-philicity with methylation.

Expressing MOMT4 in Arabidopsis Yields NovelWall-Bound Phenolics

The cell walls of monocot grasses and some dicot species, in-cluding Arabidopsis, contain significant quantities of ester-bound hydroxycinnamates, primarily p-coumarate and ferulate(Ishii, 1997; Burr and Fry, 2009). As resolved by liquid chro-matography–mass spectrometry (LC-MS; Figures 6A to 6D),expression of MOMT4 changed the profiles of those alkaline-released wall-bound phenolics. Besides two conventional phe-nolics, p-coumarate and ferulate, which were readily detected inthe cell walls of both the control and MOMT4 overexpressionlines, the bona fide phenolics were released from the walls ofMOMT4 expression lines after mild alkaline treatment of thestem cell wall residuals (Figure 6B). LC-MS/MS analysis in-dicated that one compound (P1 in Figure 6B) yielded the mo-lecular ion [M-H]2 at a mass-to-charge ratio (m/z) of 207 (Figure6C); its UV spectrum clearly showed its belonging to the cin-namic acid class of compounds that is identical to that of 4-O-methyl ferulate, the enzymatic product of MOMT4 with ferulic

Table 1. Substrate Specificity of MOMT4 on Different Phenolics

SubstrateSpecific Activity(nmol mg21 min21) Km (µM) Vmax (nmol mg21 min21)

kcat/Km

(M21 s21)

Coniferyl alcohol 66.8 6 0.7 (100)a 192.6 6 24.5b 793.0 6 3.4b 2738.7b

Sinapyl alcohol 79.6 6 0.9 (119) 68.1 6 11.1b 407.5 6 16.5b 3999.3b

Coniferaldehyde 28.7 6 0.8 (43) 6.4 6 0.5 22.0 6 0.4 2291.8Sinapaldehyde 14.6 6 0.5 (22) 3.0 6 0.5 9.3 6 0.2 2099.3P-coumaryl alcohol 5.5 6 0.1 (8.2) 190.3 6 12.0 13.7 6 0.4 48.0P-coumaraldehyde 1.9 6 0.0 (2.8) 171.0 6 20.6 3.8 6 0.2 14.6Caffeyl alcohol 1.7 6 0.0 (2.5)Ferulic acid 3.6 6 0.0 (5) 533.2 6 32.3 14.2 6 0.4 17.8Sinapic acid 1.8 6 0.0 (3) 503.6 6 12.2 20.3 6 0.2 26.9Feruloyl CoA NDc

Sinapoyl CoA NDSinapoyl glucose NDSinapoyl malate ND

The relative activity was measured from the reaction at 30°C for 30 min. Kinetic parameters were obtained from the reactions at 30°C for 10 min. Thedata represent the mean 6 SE of two replicates.aData in parentheses represent relative activity.bData obtained from radioactive assays.cND, not detectable; no data shown indicates the kinetics were not tested.




acid, and that of parental compound ferulic acid (see SupplementalFigure 7 online), suggesting P1 most likely represents 4-O-methyl-ferulate. Although the molecular mass of 207 also hints at 4-O-methyl-5-hydroxyconiferaldehyde, and sinapaldehyde, the UVspectrum of this novel wall-bound compound substantially dif-fers from those of cinnamaldehyde type of compounds (as de-picted in Supplemental Figure 7 online by the comparison withthose of sinapaldehyde and 5-hydroxyconiferaldehyde, theparental compound of 4-O-methyl-5-hydroxyconiferaldehyde).Moreover, it is known that the alkaline-releasable wall-boundphenolics are incorporated into the cell wall via ester linkages,which chemically requires cinnamic acids but not aldehydes.Together, these data suggest the presence of a novel wall-bound phenolic ester, the 4-O-methyl-ferulate, in the cell walls

of MOMT expression lines. Similarly, we identified the secondphenolic P2 (Figure 6B) that has an m/z of 237 as 4-O-methyl-sinapate (Figure 6D). The accumulation of 4-O-methylated wall-bound phenolics correlated with the expression of the MOMT4transgene; the line MOMT4-3 with the strongest gene ex-pression (Figure 3B) and lignin reduction (Table 2) amassed thehighest level of 4-O-methyl compounds, comparable to that ofthe endogenous p-coumarate or ferulate (Figure 6E).

Expressing MOMT4 Produces Novel Methanol-SolublePhenolic Esters

Profiling the methanolic extracts from the stems and leaves ofMOMT4 transgenic lines also revealed the presence of bona fide

Figure 3. Expression of MOMT4 in Arabidopsis and Histochemical Analysis of Transgenic Plants.

(A) MOMT4 overexpression cassette driven by a bean Phe ammonia lyase promoter.(B) RT-PCR analysis on the transgenes of MOMT4 and loss-function variant IEMT (E165R); both transgenes were expressed in the selected transgeniclines.(C) Morphological phenotype of wild-type, control, and MOMT4 expression plants. WT, the wild type.(D) UV autofluorescence of a stem section from the first node of a 10-week-old Arabidopsis control plant. if, interfascicular fiber; xy, xylem.(E) UV autofluorescence of a stem section from the first node of a 10-week-old MOMT4-3 transgenic line.(F) Phloroglucinol-HCl staining of a stem section from the second basal node of the control line, which indicates the total lignin in violet-red.(G) Phloroglucinol-HCl staining of a stem section from the second basal node the MOMT4-3 transgenic line.(H) Mӓule staining of a stem section from the second basal node of a control plant. The staining indicates syringyl lignin subunit in red.(I) Mӓule staining of a stem section from the second basal node of a MOMT4-3 transgenic plant.Bars = 50 µm





phenolic conjugates (Figure 7). LC-MS/MS analysis indicatedthat three novel metabolites in extracts from the 10-week-oldstems of those lines exhibited UV spectra similar to that of theenzymatic product 4-O-methyl-ferulate or sinapate, respectively,but different from those of cinnamaldehyde analogs (seeSupplemental Figure 8 online). Two compounds, the P1 and P2,yielded the molecular ion [M-H]2 at an m/z of 323 and 353, re-spectively, and a corresponding major fragment ion at m/z of 207or 237, pointing to the presence of a 4-O-methylated ferulate orsinapate residue. The loss of 116 m/z suggests their conjugationwith malate (Figures 7A to 7D). Similarly, a putative 4-O-methyl-feruloyl-1-O-glucose derivative accrued in the stems, leaves, androots of the MOMT overexpression lines (Figures 7E and 8B; seeSupplemental Figure 9 online). Although its precise structuralfeature needs to be further characterized, the MS/MS analysisrevealed that this compound yielded a daughter ion [M-H]2 of 207(Figure 7F), and its UV spectrum is largely similar to that of 4-O-methyl-ferulate but differs from that of 4-O-methyl-5-hydrox-yconiferaldehyde or sinapaldehyde (see Supplemental Figure 8online), suggesting the presence of a core structure of 4-O-methyl-ferulate in this metabolite. The difference of m/z of 162between fragment ions of 207 and 369 (Figure 7F) suggests theexistence of a Glc moiety.

The level of accumulated 4-O-methyl-feruloyl malate washigher than that of 4-O-methyl-sinapoyl ester both in the maturestems and rosette leaves of these overexpression lines (Figures8A and 8B; see Supplemental Table 3 online). As with the findingfrom previous studies (Nair et al., 2004), the conventional sina-poyl esters (sinapoyl malate and sinapoyl glucose) were pre-dominant in the rosette leaves of the control Arabidopsis,whereas, along with production of 4-O-methylated compounds,their accumulations were compromised in the leaves of theMOMT overexpression lines; however, the total amount ofquantified phenolic esters (summed for the native esters and

nascent 4-O-methyalted esters) increased up to 60% in theoverexpression lines compared with the control line (Figure 8B).In mature stems (10 weeks old), the endogenous sinapate esterswere no long detected in either the control lines or MOMToverexpression lines; in the latter, the 4-O-methylated com-pounds were the predominant esters (Figure 8A; see SupplementalTable 3 online), implying that 4-O-methylation probably alsoslows down their decomposition or conversion in the maturetissues.

Expressing MOMT4 Does Not Significantly Affect thePlant’s Growth and Development

Despite changes in the synthesis of lignin and wall-boundphenolics, the growth and developmental characteristics ofMOMT transgenic plants showed no drastic phenotypic abnor-malities, compared with either wild-type or transgenic controlplants (Figure 3C). Only at the late stages of growth (;8 weeks),the leaf senescence of MOMT overexpression lines appearedslightly earlier (Figure 3C). However, the plant height andbranching at this stage are nearly identical between control andMOMT expression lines, except the stem thickness of basalnodes of some overexpression lines was slightly decreasedcompared with the average basal node diameter of the controllines. However, the dry weight of total aboveground materialsshowed no significant difference (Table 3). Correspondingly, ata cellular level, we saw no dramatic anatomical differences in thevasculature between most of the overexpression and controllines (Figures 3G to 3J), except that slightly thinner secondarywalls were occasionally observed in a few overexpression lines.Quantifying cellulose content in the stem cell wall residues re-vealed no significant differences in cellulose deposition betweenthe control and most of the MOMT overexpression plants, al-though the amount is variable in different transgenic lines (Table

Table 2. Cell Wall Composition and Digestion Efficiency Analysis of MOMT4 Transgenic Plants

Lines

Total AcetylBromide Lignin(mg/g CWR)

Lignin Composition

Cellulose(mg/g CWR)

Digestion Efficiency(Weight Loss %)

Monomers from b-O-4–LinkedH, G, and S ConventionalLignin Units (µmol/g CWR)a

Monomers from b-O-4–LinkedH, G, and S ConventionalLignin Units (µmol/g lignin)b

S/GH G S H G S

Control 1 152.37 6 7.9 NDc 145.31 6 8.51 84.41 6 5.53 ND 1008.35 6 54.95 583.27 6 27.37 0.58 591.18 6 54.7 39.8 6 0.82Control 2 154.71 6 10.85 ND 140.90 6 2.62 76.07 6 2.62 ND 931.22 6 69.55 505.20 6 64.28 0.54 580.97 6 3.23 39.97 6 3.90Control 3 143.64 6 5.43 ND 140.05 6 18.89 72.56 6 11.99 ND 898.22 6 85.73 464.94 6 58.62 0.52 553.90 6 28.22 39.07 6 2.25MOMT4-1 128.81 6 3.25** ND 123.54 6 5.92* 59.15 6 3.89** ND 939.62 6 46.22 449.66 6 28.98 0.48 582.57 6 26.78 44.05 6 0.36*MOMT4-2 130.63 6 2.32* ND 118.57 6 4.80** 64.29 6 2.64** ND 879.73 6 48.29 476.93 6 25.88 0.54 532.12 6 8.99 44.9 6 1.76*MOMT4-3 113.47 6 1.90 ** ND 92.36 6 7.82** 51.46 6 4.95** ND 803.04 6 76.55 447.35 6 46.78 0.56 570.10 6 8.28 47.62 6 1.42**MOMT4-4 126.62 6 2.2 ** ND 113.30 6 4.29** 60.37 6 2.42** ND 898.08 6 51.02 478.48 6 29.56 0.53 582.89 6 14.11 42.99 6 1.28

The data represent the means and SE from triplicate or more biological analyses (n $ 3), except the data for lignin monomer and cellulolysis of controlplants are from duplicate analyses. Asterisks indicate significant difference compared to the average value of control lines from three independenttransgenic events: *Student’s t test, P < 0.05; **Student’s t test, P < 0.01.aYield in main lignin-derived thioacidolysis monomers (expressed in micromoles per gram of extract-free cell wall residues [CWR]) recovered fromconventional H, G, or S b-O-4–linked lignin units.bThe monomers of b were expressed in micromoles per gram acetyl bromide lignin.cND, not detectable.








2). To further examine whether expressing the artificial MOMTand producing novel phenolics would potentially impose feed-back regulation on global gene expression, we conductedtranscriptomic analysis on MOMT transgenic lines. Among themore than 22,000 genes detected, only 12 showed a moderatechange in their expression levels and none of them indicateda clear function in the central metabolic or regulatory pathways(Table 4; see Supplemental Data Set 1 online).

Expressing MOMT4 Enhances the Yield of CellWall Saccharification

We then assayed saccharification to evaluate the acceptabilityfor biodegradation of the cell walls of the MOMT transgeniclines. In keeping with their low lignin levels, exposing the cellwall residues of these transgenic plants to a commercial cellulasemixture that specifically hydrolyzes cell wall carbohydrates,

Figure 4. NMR Spectra Analysis of Lignin Structure in MOMT Overexpression Plants.

(A) Partial short-range 2D 13C–1H (HSQC) NMR spectra (aromatic and carbohydrate anomeric region) of the equal amount of solubilized total cell wallsof MOMT transgenic and control plants, after forming a gel in DMSO-d6:pyridine-d5 (4:1). The main structural units are colored to coincide with theirstructures on the right. aX1, a-D-xylopyranoside (R) [a-D-glucopyranoside (R)]; bX1, b-D-xylopyranoside (R) [b-D-glucopyranoside (R)]; U1, 4-O-methyla-D-glucuronic acid, X!1, 2-O-acetyl-b-D-xylopyranoside; X1, b-D-xylopyranoside; Gl1, (1→4)- b-D-glucopyranoside; Ar1 a-L-arabinofuranoside; Ar1(T),a-L-arabinofuranoside (T). Reducing end; T, terminal (nonreducing end).(B) Partial short-range 2D 13C–1H (HSQC) NMR spectra (aliphatic oxygenated region) of the equal amount of CELs with the main structural units coloredto coincide with their structures on the right.See Supplemental Figures 5 and 6 online for the complete set of spectra of the transgenic and control samples.




without pretreatment, raised their release of sugars by up to22% (Table 2).

DISCUSSION

Newly Engineered MOMT Mutant Variant ConfersEffective Ability in Reducing Lignin Biosynthesis

The rapid development of protein engineering strategies opensavenues for effectively manipulating plant metabolism and cre-ating desired agricultural traits. One early study by iterative geneshuffling of bacterial glyphosate N-acetyltransferase conferred anincreased tolerance of Arabidopsis, tobacco, and maize (Zeamays) to the herbicide glyphosate when the fifth iteration andbeyond mutants were expressed in planta (Castle et al., 2004).However, so far the majority of protein engineering studies havebeen performed within microorganisms, and translating from mi-crobial platforms to plants remains largely unexplored (Jez, 2011).

Figure 5. Membrane Preparation and Transport Assay.

(A) H+-ATPase activities of different membrane preparations in thepresence or absence of MgATP and/or inhibitor.(B) Transport activity of the prepared inside-out membrane vesicles toconiferyl alcohol and the 4-O-methylated coniferyl alcohol in the pres-ence and absence of ATP.Data are the means and SD of three replicates.

Figure 6. Accumulation of Wall-Bound Phenolics in MOMT Over-expression Plants.

(A) to (D) HPLC profiles of wall-bound phenolics from the cell wall res-idues of the control (A) and MOMT4 transgenic plants at the absorbanceof A330 (B). P1 is tentatively identified as 4-O-methyl-ferulic acid, and P2is tentatively 4-O-methyl-sinapic acid. The mass spectra of P1 and P2are shown in (C) and (D). The UV spectra are shown in SupplementalFigure 7 online. F, ferulic acid; st, internal standard; trans-C, trans-p-coumaric acid. mAu, milli-absorbance unit.(E) The quantification of novel 4-O-methyl wall-bound phenolic estersreleased from the cell wall of MOMT transgenic stems. The data are themeans and SE of three or four replicates. Me-F, 4-O-methyl-ferulate; Me-S, 4-O-methyl-sinapate. CWR, cell wall residue.[See online article for color version of this figure.]




Figure 7. LC-MS Analysis of Methanolic Soluble-Phenolic Compounds Accumulated in the Stems of MOMT4 Transgenic Plants.

HPLC profiles of phenolic extracts from stems of the control (A) and MOMT4 transgenic (B) plants. P1 is tentatively identified as 4-O-methyl-feruloylmalate, P2 is 4-O-methyl-sinapoyl malate, and P3 is a 4-O-methyl-feruloyl glucose derivative. The corresponding mass spectra of the compounds areshown in (C) to (F), and their UV spectra are shown in Supplemental Figure 8 online. No sinapoyl malate was detected in those mature stems. K1,kaempferol 3-O-[699-O-(rhamnosyl)glucoside] 7-O-rhamnoside; K2, kaempferol 3-O-glucoside 7-O-rhamnoside; K3, kaempferol 3-O-rhamnoside 7-O-rhamnoside; st, internal standard chrysin. mAu, milli-absorbance unit.[See online article for color version of this figure.]



Monolignol oxidative coupling, the primary process in ligninbiosynthesis, proceeds via a free radical mechanism catalyzedin situ by peroxidases or laccases. Many in vitro chemical/biophysical studies implied the importance of phenol in gen-erating active radicals for phenoxy coupling (Lloyd and Wood,1974; Dewar and David, 1980; Russell et al., 1996). Therefore,a chemical protection (masking) of the phenol (the para-hydroxyl)

would prevent radical generation and deny the derived mono-lignols the possibility of entering into the subsequent couplingprocess. This was confirmed in our study with the absolutedeprival of cross-coupling of the 4-O-methylated monolignolsin the horseradish peroxidase–H2O2-mediated polymerizationsystem (see Supplemental Figure 3 online). Moreover, our anal-ysis further revealed that the 4-O-methylated monolignol does notcompete with, nor inhibit, the oxidative coupling or polymerizationof the conventional monolignols, suggesting that 4-O-methylationremoves the capability of the modified monomer to generateradicals but does not impair oxidative enzyme activity.Despite substantial 4-O-methylation activity of the early set of

MOMT mutant variants (the single to triple mutants) to mono-lignols in vitro, they were insufficient to confer detectableeffects, when they were expressed in plants, on either ligninbiosynthesis or formation of other related phenolics. The furtheriteration, guided by the crystal structure complexed with con-iferyl alcohol, resulted in a physiologically active catalyst. Whenexpressing the mutant enzyme with four amino acid residuesubstitutions under the same transgenic strategy as for ex-pressing the triple mutants, the lignin content of the cell wallswas substantially lowered (Table 2), suggesting that the action of4-O-methyltransferase in situ indeed diminished the availabilityof monolignols for the one-electron oxidation and polymeri-zation. Compared with the triple mutant variants, the newlycreated tetra mutation variant seemingly had only a slightimprovement (approximately twofold increase) in its catalyticefficiency for methylating monolignols (see Supplemental Table1 online); however, this limited increment is sufficient to over-come the metabolic barriers, leading to reprogramming of thephenylpropanoid-monolignol biosynthetic pathways. These datasuggest that metabolic engineering of phenylpropanoid bio-synthesis requires a minimal threshold activity of the exogenousenzyme to effectively compete with the intrinsic pathway(s)/enzymes, in this case, competing against oxidative enzymes inbinding and converting available monolignols.

Expressing MOMT Diversifies Phenolic Ester Repertories

Expressing MOMT4 in Arabidopsis does not simply disrupt theendogenous monolignol biosynthetic pathway, but instead extendsthe pathway to produce “dead end” products, the para-etherifiedcompounds, for which radical polymerization is impossible.Perturbing lignin biosynthesis by the created monolignol

Figure 8. Accumulation of Soluble Phenolic Esters in MOMT Over-expression Plants.

(A) The methanol-soluble 4-O-methyl compounds accumulated in thestems of MOMT transgenic plants. Note that no conventional sinapoylesters accumulated in the mature stems of both the 10-week-old controland transgenic plants. The data are the means and SE of four replicates.FW, fresh weight; Me-FG, 4-O-methyl-feruloyl glucose derivative; Me-FM, 4-O-methyl-feruloyl malate; Me-SM, 4-O-methyl-sinapoyl malate;SG, sinapoyl glucose; SM, sinapoyl malate.(B) The methanol-soluble phenolic esters accumulated in the rosetteleaves of 7-week-old control and MOMT transgenic plants.The data represent mean and SE of four or five replicates.

Table 3. Quantitative Growth Phenotypes of MOMT4 Transgenic Plants

Genotype Height (cm) Branches Stem Diameter (mm) Dry Weight (Grams/Three Plants)

Control-1 53.75 6 1.07 5.25 6 0.25 1.71 6 0.07 1.89 6 0.11Control-2 51.92 6 1.10 5.0 6 0.33 1.43 6 0.03 2.05 6 0.13Control-3 56.67 6 1.2 5.10 6 0.15 1.65 6 0.04 2.13 6 0.07MOMT4-1 53.39 6 0.72 4.91 6 0.18 1.45 6 0.04* 2.05 6 0.11MOMT4-2 53.36 6 0.72 5.00 6 0.16 1.56 6 0.04 2.17 6 0.12MOMT4-3 52.42 6 0.77 4.83 6 0.22 1.47 6 0.04* 1.86 6 0.09MOMT4-4 53.58 6 0.93 4.83 6 0.27 1.50 6 0.04* 2.0 6 0.10

The data represent the means and SE from eight or more biological analyses (n $ 8). Asterisks indicate significant difference compared to the averagevalue of control lines from three independent transgenic events: *Student’s t test, P < 0.05.





O-methyltransferase is concomitantly associated with a rer-outing of metabolic flux to both soluble and insoluble novelphenolic compounds. Those non-natural para-methylated pro-duction of MOMT4 were found to efficiently enter into the in-trinsic phenolic ester biosynthetic pathway (Figures 6 and 7),highlighting the extreme plasticity of phenylpropanoid metabo-lism and the highly promiscuous activities of the related en-zymes that lead to a redirection in the flux of the 4-O-methylatedmetabolites, away from lignin biosynthesis.

In Brassicaceae, sinapate esters are synthesized via the phe-nylpropanoid-lignin biosynthetic pathway and are presumablystored in the vacuoles (Milkowski et al., 2004). A hydrox-ycinnamaldehyde dehydrogenase (aldehyde dehydrogenase[ALDH]) in Arabidopsis, namely REF1, reportedly can convertconiferaldehyde or sinapaldehyde to their corresponding hy-droxycinnamates that, in turn, are sequentially transformed intomalate- or choline-esters by specific glycosyltransferases andacyltransferases (Nair et al., 2004) (see Supplemental Figure 1online). MOMT4 preferentially catalyzed the 4-O-methylation ofconiferyl and sinapyl alcohols and their aldehydes in vitro. Itshowed negligible or no activity for other sinapoyl esterbiosynthetic intermediates (Table 1). These data suggestthe buildup of 4-O-methylated soluble phenolic esters mustbe de novo converted from the MOMT-produced 4-O-methyl-cinnamyl alcohols (or aldehydes). The fact that the effectivetransformation of 4-O-methylated monolignols to phenolic es-ters in transgenic Arabidopsis suggests an intrinsic cinnamylalcohol dehydrogenase (CAD) activity is recruited, which is ableto accommodate 4-O-methyl-cinnamyl alcohols and catalyzethem in a reverse reaction to the corresponding cinnamalde-hydes, and then the latter reaction further proceeded via thepromiscuous activities of REF1 and other sinapoyl ester bio-synthetic enzymes. The reversible catalytic property and sub-strate versatility of CADs in the monolignol biosynthetic pathwaywere well recognized (Kim et al., 2004) and were adopted fordetermining their functionality in vitro (Halpin et al., 1992). Thetentative pathway for transforming those compounds is pro-posed in Supplemental Figure 1 online.

It is important to note that the described strategy, using 4-O-methyltransferases to modulate lignin biosynthesis, would notbe restricted to the Brassicaceae. First, downregulation of ligninbiosynthesis by para-methylation of monolignols does not

necessarily rely on whether or not the modified monomers areable to reroute into sinapoyl ester biosynthetic pathways. Our invitro polymerization and in vivo genetic studies here demon-strate that the para-methylation of monolignols prevents theirparticipation in the oxidative coupling/polymerization process,therefore reducing lignin formation. Serendipitous production ofnovel wall-bound and soluble phenolic esters in transgenicArabidopsis represents solely an intrinsic detoxification mech-anism with respect to the versatile plasticity of phenylpropanoidmetabolism in this species. When the monolignol biosyntheticpathway is genetically disrupted, the accumulated native inter-mediates are often found either rerouted into different endoge-nous pathways or transformed into more stable or storageforms. For example, Arabidopsis mutant plants deficient in cin-namoyl-CoA reductase produced excessive levels of ferulateesters in their cell walls in stems and feruloyl- and sinapoyl-malate in the soluble fraction of stem, indicating a redirection ofaccumulated feruloyl-CoA (resulting from the disruption of cin-namoyl-CoA reductase) to wall-bound phenolic and to sina-poyl ester biosyntheses (Mir Derikvand et al., 2008); similarly,downregulation of caffeoyl CoA 3-O-methyltransferase in poplar(Populus spp) caused an accumulation of glucosides of caffeic,vanillic, and sinapic acids (Meyermans et al., 2000; Morreelet al., 2004). These phenomena suggest that plants posses anamazing versatility, recruiting their intrinsic enzymes or path-ways to evolve new metabolic capacity or detoxify biogenicproducts. Second, the Arabidopsis REF1 (ALDH) homologs wereactually also reported to exist in non-Brassicaceae species likepoplar (Nair et al., 2004). Presumably, the ubiquitous and ver-satile CAD and ALDH activities in non-Brassicaceae speciescould function in a comparable way as they do in Arabidopsis totransform 4-O-methylated monomers. Third, different specieshave developed diverse strategies to sustain their metabolichomeostasis and to transform aromatic compounds. For in-stance, poplars and other Salicaceae have evolved complicatedhydroxycinnamate pathways, by which they accumulate a vastarray of phenolic esters, and their Glc or glycosidic derivatives,such as populoside, salicin, tremuloidin, trichocarposide, etc.(Chen et al., 2010; Boeckler et al., 2011), whereas other majorgroup of gymnosperms and angiosperms are able to producemonolignol esters through acylation (e.g., coniferyl acetate) thenreduce them to a subset of volatile phenylpropenes (Koeduka

Table 4. List of Genes Whose Expression Levels Changed in MOMT Transgenic Lines

Probe Set Gene Name Annotation Transcript Level Related to Control

263046_at At2g05380 Unknown protein 2.59250277_at At5g12940 Putative protein DRT100 protein precursor 0.50248681_at At5g48900 Putative pectate lyase 0.50259331_at At3g03840 Putative auxin-induced protein 0.49265443_at At2g20750 b-Expansin 0.48250012_x_at At5g18060 Auxin-induced protein-like 0.46254066_at At4g25480 Transcriptional activator CBF1-like protein 0.45261226_at At1g20190 Putative expansin S2 precursor 0.45249614_at At5g37300 Wax ester synthase and diacylglycerol acyltransferase 0.43260727_at At1g48100 Putative polygalacturonase PG1 0.40264931_at At1g60590 Putative polygalacturonase 0.40249598_at At5g37970 SAM-dependent methyltransferase superfamily protein 0.15




et al., 2006). Therefore, the 4-O-methylated compounds in dif-ferent species, predicatively, can have sharply different meta-bolic fates, depending on the promiscuities of intrinsic enzymesor pathways being recruited. The engineered MOMT preferen-tially catalyzes monolignols (in comparison with cinnamalde-hydes), producing 4-O-methyl-cinnamyl alcohols (Table 1). Ifthere is accumulation of 4-O-methyl-cinnamaldehydes in plantaderived from the unfavorable activity of MOMT, and if there is noavailable ALDH activity to transform them to cinnamates, theycan be, expectedly, converted to the corresponding cinnamylalcohols, which is reminiscent of the classical monolignol syn-thesis, by versatile CADs, and then metabolized further by, forexample, acylation and/or glycosylation (Koeduka et al., 2006).Taken together, the collective information suggests that per-turbing lignin biosynthesis by expressing 4-O-methyltransferasewould not be limited only to Brassicaceae.

Interestingly, Arabidopsis wild-type plants accumulate noneor a negligible amount of soluble feruloyl esters (Nair et al.,2004), whereas the expression of MOMT diversified the phenolicprofiles and led to the production of a perceivable amount ofnovel feruloyl- and sinapoyl-type esters (with 4-O-methylation)that persistently accumulated in the mature tissues (Figures 7and 8). Those compounds exhibit the same or similar UV spectraas the corresponding endogenous phenolics (see SupplementalFigures 7 and 8 online); therefore, they would be predicted tooffer plants an additive defense capacity against environmentalthreats, particularly in screening out UV irradiation.

Simple phenolics are often incorporated into cell walls ofgrass species and some dicot plants and linked to different cellwall polymers via ester bonds. Those conventional wall-boundphenolics, particularly ferulate, can dimerize or polymerize witheach other or with other cell wall phenolics via free radicalmechanism, forming ester-to-ether linkages so as to cross-linkadjacent polysaccharides, lignins, and/or structural proteins(Ralph et al., 2004a; Ralph, 2010). They also potentially act asnucleation sites for lignin polymerization (Ralph et al., 2004a).Hence, the accumulation of conventional wall-bound phenolicesters might be a negative structural factor affecting cell wallbiodegradation. Expressing MOMT in planta produced ester-bound 4-O-methylated compounds (Figure 6); the 4-O-methyla-tion of phenols effectively prevents their radical cross-couplingreactions (see Supplemental Figure 3 online). Although more de-tailed analyses are required to determine their precise occur-rence within the cell walls (e.g., whether they are bound to thenoncellulosic matrix polysaccharides, lignin, or polyesters), in-corporating 4-O-methylated esters in the cell wall polymers mightafford a valuable strategy for mitigating the cross-linkage of bio-polymers, thereby improving the digestibility of the cell wall.

MOMT-Mediated Reduction of Lignin Has IndiscernibleEffect on Plant Growth and Development

Many attempts to manipulate lignin biosynthesis via the con-ventional genetic approach of regulating the monolignol bio-synthetic genes have encountered the problem of severelycompromising the plants’ fitness (Weng et al., 2008). Althoughthe underlying mechanisms of the deleterious effect might becomplicated and could be directly linked to dysfunction of the

vasculature resulting from the dramatic reduction of lignin con-tent; however, the potential accumulation of toxic metabolicintermediates due to disrupting particular monolignol bio-synthetic step(s), and/or the disturbance of the potential in-terplay of lignin biosynthesis with other biological processes,might also play a role (Li et al., 2008; Weng et al., 2008). Al-though more comprehensive stress conditions remain to beexamined, expressing MOMT, which acts on almost the laststep of monolignol biosynthesis, in planta caused little in-terference with plant growth and development under normal andcontinuous light conditions. It can therefore be speculated thatin transgenic plants, the monolignols diverted from polymeri-zation are effectively sequestered into storage form metabolitesor incorporated into the cell wall, which prevents their potentialtoxicity or their ability to act as potential feedback regulationsignals triggering global changes of gene expression as is ob-served in the downregulation of laccases (Berthet et al., 2011) orCAD (Sibout et al., 2005). Consistent with this speculation, theglobal gene expression of MOMT transgenic lines in the normalgrowth condition showed only subtle changes (Table 3).Overall, our studies offer a practical strategy to manipulate

plant lignification, without severely affecting the plant’s growthand development, by introducing an artificial enzyme to etherifythe para-hydroxyl of phenols. The approach functions in sucha way that the modified monolignols (with 4-O-methylation viathe action of the engineered enzyme) deprives the products ofparticipation in oxidative dehydrogenation and therefore di-minishes the level of monolignols available for lignin polymeri-zation. The accumulated dead end 4-O-methyl lignin monomersserendipitously enter into endogenous metabolism via the di-verse ability of plant detoxification mechanisms and the intrinsicmetabolic plasticity of plant phenylpropanoid biosynthesis.Currently, we are transferring MOMT into poplar to evaluatethe effects of expression of this enzyme on woody species’physiology.

METHODS

Saturation Mutagenesis and Mutant Library Screening

Saturation mutagenesis was performed by following the QuikChange site-directed mutagenesis strategy (Stratagene) using NNK degenerate pri-mers (Bhuiya and Liu, 2010). The QuikChange PCR products weretransferred and expressed in Escherichia coli BL21-Gold (DE3) (Stra-tagene). After induction by 0.2 mM isopropyl-b-D-1-thiogalactopyrano-side, the cells were harvested and lysed with Bugbuster solution(Novagen). The lysate was either directly used for the enzymatic assay orfurther purified to obtain recombinant proteins.

For functional screening, the reaction was conducted in 50 mM Tris-HCl, pH 7.5, containing 1 mM DTT, 300 µM coniferyl or sinapyl alcohol,500 µMSAMaugmented with (for isotope activity detection) or without (forLC-MS detection) 0.005 µCi ofS-adenosyl-L-Met-(methyl-14C), and 20mLof lysate or purified protein. Reactions were incubated for 30 min at 30°C.The steady state kinetics of MOMT variants were determined using dif-ferent concentrations of selected substrates at a fixed concentration of500 µM SAM. Reactions proceeded for 10 min at 30°C. The Vmax and Km

were determined by nonlinear regression analysis of the velocity con-centration data fit to the Michaelis-Menten equation. When examiningsubstrate specificity, purified MOMT4 (T133L-E165I-F175I-H169F) wasused in the assay.





Crystallization and Structure Determination of MOMT3

Crystals ofMOMT3 (T133L-E165I-F175I) in complex with SAH and coniferylalcohol were obtained through the hanging-drop vapor diffusion method.Diffraction data (see Supplemental Table 2 online) were collected fromsingle crystals at the X29- and X25-beamlines of National Synchrotron LightSource. The initial structure model was determined by the molecular re-placement using COMT structure (Protein Data Bank code 1KYW) (Zubietaet al., 2002) via the Phaser program in CCP4i suite. The built model wasrefined using CNS 1.1 and Refmac5 (Vagin et al., 2004). The final model wasbuilt based on composite omit map to avoid structural bias.

In Vitro Polymerization

The 4-O-methylated coniferyl or sinapyl alcohol was produced in theenlarged reaction using MOMT4 enzyme. The enzymatic products werepurified by Strata-X polymeric sorbents column (Phenomenex). At-tempted in vitro polymerization and product examination by LC-MS wasperformed based on the method described (Bhuiya and Liu, 2010). Forcoincubation of monolignol and 4-O-methylated monolignol, the coniferylalcohol was fixed at the concentration of 5 mM, and 0.5 to 50 mM 4-O-methyl-coniferyl alcohol was mixed into the reaction mixture containing136 ng/mL horseradish peroxidase and 1.14 mM H2O2. The reactionswere allowed to proceed for 5 min and then stopped by addition of equalvolume of 50% acetonitrile. The products were separated by HPLC using0.2% acetic acid (A) with an increasing concentration gradient of ace-tonitrile containing 0.2% acetic acid (B) for 0 to 2 min, 5%; 2 to 8 min, 5 to39%; 8 to 10min, 39 to 45%; 10 to 14min, 45 to 50%; and 14 to 17min, 50to 100%; at a flow rate of 1 mL/min. The peak areas of the major oxidativedimeric products were summed to quantify the polymerization efficiency.

RNA Extraction and RT-PCR

Stems of MOMT4 transgenic and control plants grown in the same growthchamber were collected at stage 6.2 (Boyes et al., 2001). For each line, wepooled the entire stems of three individual plants to constitute onesample. Samples were immediately frozen in liquid nitrogen and stored at280°C until use for total RNA extraction.

Total RNA was extracted with the Qiagen RNeasy plant mini kitaccording to the manufacturer’s instructions. RNA samples were quan-tified with a Nanodrop spectrophotometer (ND-1000; Labtech). Reversetranscription was performedwith 3mg of total RNA andM-MLV (Promega)according to the manufacturer’s instructions. TheMOMT4 transgene wasdetected using primers 59-GCTCACGGCTACCGACAAGG-39 and 59-GCAATGCTCATCGCTCCAGT-39. The internal standard ACTIN2 wasamplified with primers 59-GGTAACATTGTGCTCAGTGGTGG-39 and 59-CTCGGCCTTGGAGATCCACATC-39.

In Vitro Transport Assay

Preparation of plasma membrane inside-out vesicles, their quality ex-amination, and the uptake assay on coniferyl alcohol and 4-O-methylatedconiferyl alcohol were performed essentially as described byMiao and Liu(2010).

Briefly, anArabidopsis thalianamicrosomal fraction was prepared from;4-week-old plants of the wild type (Columbia-0) according to Palmgrenet al. (1990). Plasma membranes were then purified by partitioning themicrosomal fraction into an aqueous polymer two-phase system asdescribed (Larsson et al., 1994). The final upper phase containing theright-side-out plasmamembranes was pelleted and resuspended in 5mMpotassium phosphate buffer, pH 7.8, containing 0.33 M Suc, 5 mM KCI,1 mM DTT, and 0.1 mM EDTA (buffer A). The plasma membrane vesicleswere then mixed with the detergent Brij 58 (Sigma-Aldrich) to a finalconcentration of 0.05% (w/v) and KCl to 50 mM, together with four cycles

of freezing-thawing, to produce the sealed, inside-out vesicles as de-scribed (Johansson et al., 1995). The inverted plasma membrane vesicleswere pelleted and gently resuspended in 10 mM MOPS-KOH buffer, pH7.5, containing 0.33 M Suc, 0.1 mM EDTA, 1 mM DTT, and 13 proteaseinhibitor cocktail (Sigma-Aldrich). The quality of the prepared plasmamembranes was monitored by measuring the vanadate-inhibitedMg2+-ATPase activity by an ATPase assay kit, according to the manu-facturer’s instructions (Innova Biosciences).

The uptake assay was performed at 25°C for 30 min in a 500 mL assaymixture containing 25 mM Tris-MES, pH 8.0, 0.4 M sorbitol, 50 mM KCl,5 mMMg-ATP, 0.1% (w/v) BSA, and 100 mM of phenolic substrates. Mg-ATP was omitted from the nonenergized controls. Assays were initiatedby the addition of membrane vesicles (100 mg of protein) and brief agi-tation. After incubation, the mixtures were subjected to vacuum filtrationthrough prewetted nitrocellulose membrane filters (0.22-µm pore di-ameter; Millipore). After thorough washing, the filters were then extractedby 50% (v/v) methanol in an orbital shaker. The methanolic extracts wereanalyzed by HPLC. Quantification wasmade based on the standard curveof coniferyl alcohol.

Overexpression of MOMT4 in Arabidopsis

The open reading frames of MOMT4 and a loss-of-function mutant(E165R, as the control) (Bhuiya and Liu, 2010) were integrated into a binaryvector pMDC32-PAL-GW that was constructed by replacement of the35S promoter of pMDC32, a gateway destination vector (Curtis andGrossniklaus, 2003), by the PAL2 promoter. The constructs weretransferred into Arabidopsis wild type (Columbia-0) mediated by Agro-bacterium tumefaciens via the floral dip method (Clough and Bent, 1998).Arabidopsis plants were grown in a growth chamber at 22°C with 60%relative humidity under a 14-h-light (110mmol m21 s21)/10-h-dark regime.The transgenic and control T2 or T3 generation plants were grown side byside for lignin and phenolic analysis.

Histochemical Analysis

Histochemical analysis was performed using 30-µm-thick sections fromthe same position of second basal node from 8-week-old stems of controland MOMT4 transgenic plants following the standard protocols forphloroglucinol (Wiesner) and Mӓule staining (Jensen, 1962; Nakano andMeshitsuka, 1992).

Analysis of Wall-Bound Phenolics and Lignin

Arabidopsis stems were harvested from 10-week-old plants at stage of9.70 (Boyes et al., 2001). Stems from at least five individual plants for eachtransgenic line were grouped and ground into powder under liquid ni-trogen. The powder was extensively extracted with 70% ethanol at 65°Cfor 3 h and repeated three times and then the residuals were treated withacetone at room temperature overnight. The extractive free residuals weredried at 45°C and then ball-milled into a fine powder. The wall-boundphenolics were extracted with 2 N NaOH in the dark at 37°C for 16 h andanalyzed as described (Gou et al., 2008). Total lignin was quantified by theacetyl bromide method (Chang et al., 2008). To analyze the monomericcomposition of lignin, we followed the thioacidolysis method (Rolandoet al., 1992). Quantification of the corresponding monomers was donewith a gas chromatography–flame ionization detector (Agilent) after anappropriate calibration relative to the tetracosane internal standard. Thestatistical analyses on the obtained data sets were performed witha Student’s paired t test, using two-tailed distribution and two-sampleunequal variances.

For whole-cell wall NMR analysis, the ball-milled extractive-freeresiduals (55 mg) were mixed with 4:1 DMSO-d6/pyridine-d5 (500 mL) toform gel samples (Kim and Ralph, 2010). For a more complete and in-



Wadud Bhuiya

Wadud Bhuiya

depth structural characterization of lignin, cell wall residuals were di-gested by Cellulysin (cellulase; Calbiochem) to obtain CEL. Tenmilligramsof CELs were dissolved in 500 mL of DMSO-d6. 2D-HSQC NMR spectrawere acquired on a Bruker Biospin Avance 500 MHz spectrometer fittedwith a cryogenically cooled 5-mm triple-resonance gradient probe opti-mized for 1H and 13C observation with inverse geometry as previouslydescribed (Kim and Ralph, 2010).

Cellulose Quantification and Cellulolysis

Cellulose content was measured essentially according to the Updegraff’smethod (Updegraff, 1969) at 620 nm on ball-milled cell wall materials. Theefficiency of saccharification of cell wall biomass was determined usinga described method (Sibout et al., 2005).

Phenotypic Analysis of MOMT4 Transgenic Plants

Eight-week-old plants between stages of 6.50 and 6.90 (Boyes et al.,2001) were used for phenotypic analysis. The shoot length of the mainstem was measured by ruler as the height of the individual plant. The baseof the main stem was measured using a Vernier caliper. The abovegroundparts of three plants were sealed in paper bags and dried to constantweight (;5 d) in an oven at 60°C.

Analysis of Soluble Phenolics

Roots, stems, leaves, and seeds of transgenic plants were ground intopowder in liquid nitrogen andwere extractedwith 80%methanol containing25 µM chrysin as the internal standard at 4°C overnight. The extracts wereprofiled by LC-MS and characterized byMSn analysis. Quantification of thedetected compounds was based on the standard curves of ferulic acid andsinapic acid that were made in the same HPLC runs.

Accession Numbers

The atomic coordinates and structure factors, MOMT3-coniferyl alcohol-SAH with code 3TKY, have been deposited in the Protein Data Bank,Research Collaboratory for Structural Bioinformatics (Rutgers University,NewBrunswick, NJ) (http/www.rcsb.org/). Sequence data from this articlecan be found in the Arabidopsis Genome Initiative or GenBank/EMBLdatabases under the following accession numbers: JX287369 (MOMT4)and AT3G18780 (ACTIN2).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. The Scheme of Simplified Phenylpropanoid-Lignin-Phenolic Ester Biosynthetic Pathways.

Supplemental Figure 2. Amino Acid Residues Directly in Contactwith, or Proximate to, the Bound Coniferyl Alcohol in the MOMT3Active Site.

Supplemental Figure 3. In Vitro Polymerization of ConventionalMonolignols and 4-O-Methylated Monolignols.

Supplemental Figure 4. Coincubation of Conventional Monolignolwith 4-O-Methylated Monolignol in the in Vitro Polymerization System.

Supplemental Figure 5. HSQC NMR Spectra (dc/dh 0 to 135/0 to 8.0ppm) of the Total Cell Wall Samples of MOMT4 Transgenic andControl Plants after Gelling in DMSO-d6:Pyridine-d5 (4:1).

Supplemental Figure 6. Partial Short-Range HSQC NMR Spectra(Aromatic Region) of Cellulolytic Enzyme Lignins Isolated from CellWalls of MOMT4 Transgenic and Control Plants.

Supplemental Figure 7. The UV Spectra of Novel Wall-BoundPhenolic Esters and the Related Phenolic Standards.

Supplemental Figure 8. The UV Spectra of Novel Soluble PhenolicEsters and the Related Phenolic Standards.

Supplemental Figure 9. LC-MS Analysis on Novel MethanolicSoluble-Phenolic Compounds Accumulated in the Root of MOMT4Transgenic Plants

Supplemental Table 1. The Kinetic Parameters of MOMT Variants forMonolignols.

Supplemental Table 2. Crystallographic Data Sheet for the Structureof MOMT3 Mutant Variant.

Supplemental Table 3. Quantification of Soluble Phenolics in 10-Week-Old Stems of MOMT4 Transgenic Plants.

Supplemental Data Set 1. Complete Data Set of Transcriptome Studyof MOMT Transgenic Line by cDNA Microarray Analysis.

ACKNOWLEDGMENTS

We thank Eran Pichersky (University of Michigan) for donating the Clarkiabreweri IEMT clone. We also thank John Shanklin and William Studier(Brookhaven National Laboratory) for their valuable discussions on thiswork. This work is supported by the Division of Chemical Sciences,Geosciences, and Biosciences, Office of Basic Energy Sciences of theU.S. Department of Energy (DOE) through Grant DEAC0298CH10886(BO-147 and 157) to C.-J.L. Transport measurement (by Y.M.) wassupported in part by National Science Foundation through Grant MCB-1051675. J.R.P. was funded by a Spanish Ministry of Education (post-doctoral fellowship); J.R., J.R.P., and H.K. (NMR analysis) were funded inpart by the DOE’s Great Lakes Bioenergy Research Center (Grant BERDE-FC02-07ER64494). The use of x-ray beamlines at X25 and X29 ofNational Synchrotron Light Source is supported by the DOE, Office ofBasic Energy Sciences, under Contract DEAC0298CH10886.

AUTHOR CONTRIBUTIONS

C.-J.L., K.Z., and M.-W.B. designed the research. K.Z, M.-W.B, andC.-J.L. performed biochemical, structural, and genetic experiments. Y.M.performed the transport assay, and J.R.P. and H.K. performed NMRanalysis. C.-J.L., J.R, K.Z., and M.-W.B. analyzed data. C.-J.L., J.R.,K.Z., and M.-W.B. wrote the article. All authors contributed to theeditorial efforts.

Received June 6, 2012; revised June 25, 2012; accepted July 10, 2012;published July 31, 2012.

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Wadud Bhuiya

OH

HO

OH

HO

OH

HO

HOOH

HO

H

HO

OH

HO

OMe

OHO

H

HO

OMe

OHO

H

HO

OMe

O

SCoA

HO

O

SCoA

HO

O

OMe

OH

O

NH2

OH

HO

O

OH

O

SCoA

HO

O

OH

L-phenylalanine

Cinnamic acid

p-Coumaric acid

p-Coumaroyl-CoA

p-Coumaraldehyde

p-Coumaryl alcohol

Caffeoyl-CoA Feruloyl-CoA

Coniferaldehyde 5-OH-coniferaldehyde Sinapaldehyde

Coniferyl alcohol 5-OH-coniferyl alcohol

Sinapyl alcohol

Guaiacyl lignin

Cytoplasm

Syringyl ligninp-Hydroxyphenyl lignin

Cell wall

PAL

C4H

4CL

CCR

CAD

POXLAC

CAD CAD

HCT C3’H HCT CCoAOMT

CCRCald5H COMT

COMTCald5H

POXLAC

POXLAC

OH OH

4-O-methyl-coniferyl alcohol

4-O-methyl-sinapyl alcohol

4-O-methyl-coniferaldehyde

H

MeO

OMe

MeO

O

H

OMe

O

4-O-methyl-sinapaldehyde

Ferulic acid4-O-methyl-ferulic acid 4-O-methyl-sinapic acid

4-O-methyl-feruloyl glucose

Sinapic acid

Sinapoyl glucose 4-O-methyl-sinapoyl glucose

4-O-methyl-feruloyl malate Sinapoyl malate4-O-methyl-sinapoyl malate

CAD? CAD?MOMT

MOMT MOMT

MOMT

ALDH

(ref1)

ALDH

(ref1)

ALDH

(ref1)

SGT

SMT SMT SMT

ALDH

(ref1)

Vacuole

COMTF5HSGT SGT

Supplemental Figure 1. The scheme of simplified phenylpropanoid-lignin-phenolic ester biosynthetic pathwaysArrows in black illustrate monolignol biosynthetic pathway. Arrows in magenta indicate the native sinapoyl malate pathway. Arrows in green indicate the putative pathways leading to the formation of novel phenolic esters after expression of MOMT. Dashed line arrows imply the putative steps. PAL: phenylalanine ammonia lyase, C4H: Cinnamic acid 4-hydroxylase, 4CL: 4-Hydroxycinnamoyl CoA ligase, HCT: Hydroxycinnamoyl CoA: shikimate/quinate hydroxycinnamoyl-transferase, C3΄H: p-Coumaroylshikimate 3΄-hydroxylase, CCoAOMT: Caffeoyl CoA O-methyltransferase, CCR: Cinnamoyl CoA reductase, Cald5H/F5H: Coniferaldehyde/ferulate 5-hydroxylase, COMT: Caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase, CAD: (Hydroxy)cinnamyl alcohol dehydrogenase, POX: Peroxidase, LAC: Laccase. ALDH: Aldehyde dehydrogenase, SGT: Sinapate glucosyltransferase, SMT: sinapoylglucose:malate sinapoyltransferase.

OMe

MeO

MeO

OMe

MeO

OMeOMe

MeO

MeO

OMe

Supplemental data. Zhang et al. (2012). Plant Cell 10.1105/tpc.111.101287

A134L133

I175

V138Y326F179

L139

H169

F157

I165

F166 N164

H272 M323

F130

N186

T319M183

L322

A325

H173

Y168

Y161

N327


Supplemental Figure 2. Amino-acid residues directly in contact with, or proximate to the bound coniferyl alcohol in the MOMT3 active site.The residues targeted for the iterative saturation mutagenesis were F130, A134, V138, L139, F157, Y161, N164, F166, Y168, H169, H173, F179, M183, N186, T319, L322, M323, A325, Y326, and N327.

191297

309MS2 (338)

402

387372205

181

166

MS2 (417)

59

4x104

195 327

375G( -O-4)G

2x104

327

195179165

MS2 (375)

1x104

2x104

339

327357

G( -5)G

1x105

136

151

176 313 342

MS2 (357)

1x104

59 117137159 321

342

357G( - )G

1x104

4x103

417S( - )S

4x105

4x104

Mw:358

OHO

OMeHO

OH

OMeB

A

191

327

339

Mw:418

O

O

OH

OMe

HO

MeO

205

181

166

B

A

OMe

OMeMw:376

HOO

HOOH

OH

OMe

O

4

A

B

A

151

179

5

AHRP, H2O2

80

60

40

20

0

10 12 14 16 18 20

OMe

OHO

OH

5OMe

OH

G( -5)G

G( - )GG( -O-4)G

OMe

OH

OH

80

60

40

20

0

10 12 14 16 18 20

OMe

OH

OMe

HRP, H2O2 XC

OMe

OH

OMeMeO

HRP, H2O2 X80

60

40

20

010 12 14 16 18 20

D

Retention time (min)

HOOMe

O

O

OHOMe

OMe

MeO

B40

30

20

10

0

10 12 14 16 18 20

OMe

OH

OHMeO

S( - )S

HRP, H2O2

Mw:358

O

O

OH

OMe

HO

MeO

176

151

136

B

A

Abs

orba

nce

at 2

80 n

m (m

AU

)

Supplemental Figure 3. In vitro polymerization of conventional monolignols and 4-O-methylated monolignols. (A) LC-MS profile of the reaction of coniferyl alcohol incubated with horseradish peroxidase and H2O2 (solid line), or with buffer alone as the control (dashed line). The profile shows a predominant dimer G(β–5)G and the minor products of G(β–O–4)G and G(β–β)G. The corresponding ESI-mass spectra of the dimeric products are shown. (B) LC-MS profile of the reaction of sinapyl alcohol incubated with horseradish peroxidase and H2O2 (solid line), or with buffer alone as the control (dashed line). The profile identifies a dimer S(β–β)S. Its tandem ESI-mass spectra are shown. (C) LC-MS profile of the reaction of 4-O-methyl-coniferyl alcohol incubated with horseradish peroxidase and H2O2 (solid line), or with buffer alone (dashed line). As expected, no dimers or oligomers were detected.(D) LC-MS profile of the reaction of 4-O-methyl-sinapyl alcohol incubated with horseradish peroxidase and H2O2 (solid line), or with buffer alone (dashed line). As expected, no dimers or oligomers were detected.


6 8 10 12 14 16

0

2

4

6

8

18

10 CA + H2O2 + Peroxidase

CA + 4-O-methyl-CA + H2O2 + Peroxidase

6 8 10 12 14 16

0 2 4 6 8

18

10 12

Retention time (min) 6 8 10 12 14 16

0 2 4 6 8

18

10 12

0

20

40

60

80

100

0 1 2.5 5 10 25 50

Rel

ativ

e Pr

oduc

tion

(%)

Concentration of 4-O-methyl coniferyl alcohol (µM)

G(ȕ-O-4)G

G(ȕ-5)G

Coniferyl alcohol St

4-O-methyl-CA St

(A)

(B)

G(ȕ-O-4)G

G(ȕ-5)G

Supplemental Figure 4. Co-incubation of Conventional Monolignol with 4-O-Methylated Monolignol in the In Vitro Polymerization System. (A) HPLC profiles of the reactions incubating coniferyl alcohol alone (red line), or both coniferyl alcohol and 4-O-methyl-coniferyl alcohol (blue line) with horseradish peroxidase and H2O2. No difference was discernible in the production of oligomers from the two sets of reactions. CA: coniferyl alcohol. (B) Examination of potential inhibition of 4-O-methyl-coniferyl alcohol on oxidative coupling of coniferyl alcohol. The production of major dimers *�ȕ–5)G and *�ȕ–O–4)G was quantified and compared to that in the reaction of coniferyl alcohol alone (which was set as 100%). The data are the mean and SD of two replicates.

Supplemental data. Zhang et al. (2012).Plant Cell 10.1105/tpc.111.101287

Control 1

}

6 4 2

20

40

80

100

60

120

}

6 4 2

20

40

80

100

60

120 }

6 4 2

20

40

80

100

60

120

}

6 4 2

20

40

80

100

60

120

Control 2

MOMT4-3

MOMT4-1

Supplemental Figure 5. HSQC NMR spectra (δC/δH 0-135/0-8.0 ppm) of the cell walls of MOMT4transgenic and control lines after forming a gel in DMSO-d6:pyridine-d5 (4:1).


Supplemental Figure 6. Partial short-range 13C-1H(HSQC) spectra (aromatic regions) of CELs isolated from the cellwalls of two control and MOMT transgenic lines.

}

7.5 7.0 6.5

105

110

115

120

125

130

Control 1

}

MOMT4-3

7.5 7.0 6.5

105

110

115

120

125

130

}

Control 2

7.5 7.0 6.5

105

110

115

120

125

130

}

MOMT4-1

7.5 7.0 6.5

105

110

115

120

125

130


nm 200 250 300 350 400 450 500 550

0

5

10

15

20

25 Sinapaldehyde P1

nm 200 250 300 350 400 450 500 550 600

0

5

10

15

20

25 5-Hydroxyconiferaldehyde P1

10

15

20

25

nm 200 250 300 350 400 450 500 550 600

0

5

Ferulic acid P1

nm 200 250 300 350 400 450 500 550

0

5

10

15

20

25 4-O-methyl-ferulic acid P1

nm 200 250 300 350 400 450 500 550

0

5

10

15

20

25 4-O-methylsinapic acid P2

30

600

Supplemental Figure 7. The UV Spectra of Novel "Wall-Bound" Phenolic Esters and the Related Phenolic Standards. The spectrum of P1 in Figure 6 is identical to that of the enzymatic product 4-O-methyl-ferulic acid and similar to its parental compound ferulic acid, but differs from those aldehyde analogues; similarly the spectrum of P2 is identical to that of enzymatic product 4-O-methyl-sinapic acid.


nm 200 300 400 500 600

0 10 20 30 40 50 60

Ferulic acid P1

nm 200 300 400 500 600

0 10 20 30 40 50 60

4-O-methylferulic acid P1

nm 200 300 400 500 600

0 10 20 30 40 50 60

5-hydroxyconiferaldehyde P1

nm 200 300 400 500 600

0 10 20 30 40 50 60

Sinapaldehyde P1

nm 200 300 400 500 600

0 10 20 30 40 50

4-O-methylsinapic acid P2

nm 200 300 400 500 600

0

50 100 150 200 250

Ferulic Acid P3 P3

100

150

200 250

nm 200 300 400 500

0

50

600

4-O-methyl ferulic acid

nm 200 300 400 500 600 0

50

100

150

200

250 P3 Sinapaldehyde

nm 200 300 400 500 600

250

0 50

100 150 200 5-Hydroxyconiferaldehyde

P3

Supplemental Figure 8. The UV Spectra of Novel Soluble Phenolic Esters and the Related Phenolic Standards. The spectrum of P1 and P3 in Figure 7 is similar to those of the enzymatic product 4-O-methyl-ferulic acid and its parental compound ferulic acid, but differs from those of aldehyde analogues; the spectrum of P2 is similar to that of enzymatic product 4-O-methyl-sinapic acid.


Supplemtal Figure 9. LC-MS analysis on novel methanolic soluble-phenolic compounds accumulated in the root of MOMT4 transgenic plants. (A) HPLC profiles of phenolic extract from roots of control plants, and (B) MOMT4 transgenic plants. The major novel compound accumulated in MOMT4 expressionplant is the 4-O-methyl-feruloyl glucose derivative; its corresponding MS spectra are shown in (C).

0

4-O-methyl-feruloyl glucose derivative

)uA

m( mn 033 ta ecnabrosb

A

A

Retention time (min)

B

100

200

300

6010 15 20 25 30 35 40 45 50 55

400

100

200

300

6010 15 20 25 30 35 40 45 50 55

400

416.0

463.3

577.1

207.3

369.1-MS2 (416.4)

MS -

-162 u

C

ecnadnubA

2x105

4x105

6x105

0.5x105

1x105

100 300 500 700 900 m/z

100 300 500 700 900 m/z

0



Supplemental Table 1. The Kinetic parameters of MOMT variants for monolignols.

The data represent the mean ± S.E. of two replicates.

Protein Km (µM)

Vmax (nmol. mg -1.min -1)

Kcat/Km (M-1 s-1)

Coniferyl alcohol

IEMT wild type 1591 ± 182 42 ± 3.5 17.6

T13M-E165F 546.4 ± 62.2 352.7 ± 19.2 430.3

T133L-E165F 279.9 ± 18.3 117 ± 2.9 278.6

T133L-E165I-F175I 279.2 ± 48.2 556.2 ± 35.6 1327.9

T133L-E165I-F175I-Y161W 196.3 ± 33.6 583.8 ± 36.9 1986.1

T133L-E165I-F175I-Y161F 196.5 ± 21.2 642.2 ± 24.2 2172.2

T133L-E165I-F175I-F166W 117.6 ± 15 496.6 ± 8.2 2825.7

T133L-E165I-F175I-H169F 192.6 ± 24.5 793 ± 32.4 2738.7

T133L-E165I-F175I-H169M 219.7 ± 27 629 ± 26.2 1905.7

T133L-E165I-F175I-A325V 235.9 ± 49.8 446.2 ± 32.7 1259.7

T133L-E165I-F175I-F166W-H169W 127.2 ± 8.1 392.1 ± 9.4 2054.7

T133L-E165I-F175I-F166W-T135N 166.9 ±10.9 431.3± 11.7 1722.5

T133L-E165I-F175I-Y326F-N327V 143.5 ± 12.8 474.5 ± 12.4 2204

Sinapyl alcohol

IEMT wild type 1495 ± 214.4 44.6 ± 3.9 19.9

T13M-E165F 326 ± 59.5 84.7 ± 6.2 173.2

T133L-E165F 381.9 ± 32.3 102.7 ± 3.7 179.2

T133L-E165I-F175I 119.6 ± 15.5 274.7 ± 9.5 1527.5

T133L-E165I-F175I-Y161W 218.7 ± 19.1 259.1 ± 7.6 788.3

T133L-E165I-F175I-Y161F 205.6 ± 25 289.2 ± 12.3 937.6

T133L-E165I-F175I-F166W 47.1 ± 8.6 146.6 ± 5.3 2084.7

T133L-E165I-F175I-H169F 68.1 ± 11.1 407.5 ± 16.5 3999.3

T133L-E165I-F175I-H169M 88.9 ± 10.4 369.7 ± 11.0 2771.1

T133L-E165I-F175I-A325V 96.9 ± 6.9 214 ± 3.8 1470.5

T133L-E165I-F175I-F166W-H169W 67.6 ± 12.9 160.5 ± 11.9 1582.6

T133L-E165I-F175I-F166W-T135N 54.5 ± 14.2 115 ± 8.6 1406.5

T133L-E165I-F175I-Y326F-N327V 298.3 ± 29.2 301 ± 11.2 672.6


Supplemental Table 2. Crystallographic data sheet for MOMT3-coniferyl alcohol-SAH structure

a Number is parentheses refer to the highest shell. b Rsym = |Ih – <Ih>|/Ih, where <Ih>| is the average intensity over symmetry equivalent reflections. c Rcryst = �_)obs – Fcalc|/ �)obs, where summation is over the data used for refinement, Rfree was calculated using 5% of data excluded from refinement.

Data collection Space group P21 Cell dimensions a, b, c (Å) 65.86, 152.16, 68.18 Į��ȕ��Ȗ��Û) 90.00, 94.92, 90.00 Resolution range (Å)a 50-2.4 (2.47-2.40) Observations 291,566 Unique reflections 52,117 Completeness (%)a 100 (99.9) Redundancy a 5.6 (5.1) I/ı a 26.1 (2.0) Rsym (%) a,b 13.0 (50.4) Refinement Rcryst/Rfree (%)c 19.3 (26.1) No. of atoms Protein 10743 Water molecules 166 SAH 104 Phenolic substrate 52 RMSD Bonds (Å) 0.008 Angles (Û� 1.4 Average B factors Protein (Å2) 20.8 water (Å2) 19.1 SAH (Å2) 18.7 Phenolic substrate (Å2) 80.6


Supplemental Table 3. Quantification of soluble phenolics in ten-week-old stems of MOMT4 transgenic plants a

Phenolics Control MOMT-1 MOMT-2 MOMT-3 MOMT-4

4-O-methyl-feruloyl malateb ND 619.6 ± 21 411 ± 26.1 770.5 ± 53.7 384.6 ± 57

4-O-methyl-sinapoyl malate ND 153 ± 4.6 127.3 ± 6.4 316.5 ± 10.1 192.8 ± 11

4-O-methyl-feruloyl glucose ND 57.8 ± 1.7 17.2 ± 2.8 41.8 ± 8.3 13.8 ± 2.7

Sinapoyl malate ND ND ND ND ND

Feruloyl malate ND ND ND ND ND

Kaempferol 3-O-[6''-O-

(rhamnosyl) glucoside]-7-O-

rhamnoside

204.7 ± 66.2 269.5 ± 52.1 147 ± 30.6 301.5 ± 38.2 180.3 ± 32.7

Kaempferol 3-O-

rhamnoside-7-O-glucoside

268.2 ± 45 231.1 ± 57.2 112.4 ± 27.5 251.8 ± 32.7 150.7 ± 31.3

Kaempferol 3-O-rhamnoside

7-O-rhamnoside

608.3 ± 112.1 551.6 ± 84.4 330.2 ± 53 617.1 ± 62.5 368 ± 53.7

a The data represent the means and standard errors from four or five analyses (n�� bThe quantity of the soluble compounds are expressed in nanomole per gram fresh weight stems

ND, Not detectable

DOI 10.1105/tpc.112.101287; originally published online July 31, 2012;Plant Cell

and Chang-Jun LiuKewei Zhang, Mohammad-Wadud Bhuiya, Jorge Rencoret Pazo, Yuchen Miao, Hoon Kim, John Ralph

ArabidopsisNovel Metabolic Capability in -Methyltransferase Depresses Lignin Biosynthesis and ConfersOAn Engineered Monolignol 4-

This information is current as of July 31, 2012

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an engineered monolignol 4-o-methyltransferase depresses lignin biosynthesis and confers novel...

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